research article the impacts of swimming exercise on...

Research ArticleThe Impacts of Swimming Exercise on HippocampalExpression of Neurotrophic Factors in Rats Exposed toChronic Unpredictable Mild Stress

Pei Jiang Rui-Li Dang Huan-De Li Li-Hong ZhangWen-Ye Zhu Ying Xue and Mi-Mi Tang

Institute of Clinical Pharmacy amp Pharmacology Second Xiangya Hospital Central South University Changsha 410011 China

Correspondence should be addressed to Huan-De Li lihuande1953126com

Received 24 June 2014 Accepted 14 August 2014 Published 27 October 2014

Academic Editor David Mischoulon

Copyright copy 2014 Pei Jiang et alThis is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Depression is associated with stress-induced neural atrophy in limbic brain regions whereas exercise has antidepressant effectsas well as increasing hippocampal synaptic plasticity by strengthening neurogenesis metabolism and vascular function Akey mechanism mediating these broad benefits of exercise on the brain is induction of neurotrophic factors which instructdownstream structural and functional changes To systematically evaluate the potential neurotrophic factors that were involved inthe antidepressive effects of exercise in this study we assessed the effects of swimming exercise on hippocampal mRNA expressionof several classes of the growth factors (BDNF GDNF NGF NT-3 FGF2 VEGF and IGF-1) and peptides (VGF and NPY) in ratsexposed to chronic unpredictable mild stress (CUMS) Our study demonstrated that the swimming training paradigm significantlyinduced the expression of BDNF and BDNF-regulated peptides (VGF and NPY) and restored their stress-induced downregulationAdditionally the exercise protocol also increased the antiapoptotic Bcl-xl expression andnormalized theCUMSmediated inductionof proapoptotic Bax mRNA level Overall our data suggest that swimming exercise has antidepressant effects increasing theresistance to the neural damage caused by CUMS and both BDNF and its downstream neurotrophic peptides may exert a majorfunction in the exercise related adaptive processes to CUMS

1 Introduction

Depression is a debilitating and widely distributed disorderwhich is associated with exposure to stressful life eventsStudies of chronic stress in animal models and postmortemtissues from depressed patients demonstrated that reducedsize of limbic brain regions that regulate mood and cognitionand decreased neuronal synapses in these brain areas maycontribute to the pathogenesis of depression [1] There isemerging evidence that exercise has antidepressant effectswhereby promoting neurogenesis and inhibiting neurode-generation [2] Although exercise seems to have therapeuticand preventive effects on the course of depression theunderlying mechanisms remain elusive It has been proposedthat the key mechanism mediating the broad benefits ofexercise on the brain is induction of neurotrophic fac-tors which instruct downstream structural and functional

changes [3] The protective effects of exercise from chronicstress have been best-studied in the hippocampus whereexercise increased synaptic plasticity and neurotrophic fac-tors expression Previous studies indicate that exercise canpromote hippocampal neurotrophic cascades and enhanceneural survival differentiation connectivity and plasticitywhile stress shows the opposite effects which indicates apotential mechanism for exercise to alleviate stress [4]

Brain-derived neurotrophic factor (BDNF) is the mostabundantly expressed neurotrophin in the mature centralnervous system and supports the survival of many types ofneurons A number of animal studies have documented thatthe exposure to chronic stress can result in decreased BDNFexpression in hippocampus [5] Conversely both antidepres-sant treatment and exercise can enhance hippocampal BDNFstatus [6 7] The neuropeptide VGF and neuropeptide Y(NPY) have been implicated in the actions of BDNF and

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 729827 8 pageshttpdxdoiorg1011552014729827

2 Evidence-Based Complementary and Alternative Medicine

both of which can be induced by BDNF and antidepressants[8 9] Accumulating evidence has shown that BDNF wasimplicated in the pathophysiology of depression and theantidepressant action of exercise Nevertheless the role ofits downstream neuropeptides and other neurotrophic fac-tors remains unclear Other neurotrophins including nervegrowth factor (NGF) glia cell-derived neurotrophic factor(GDNF) and neurotrophin-3 (NT-3) are also importantfactors for regulation of neuroplasty and were implied toplay a role in the neurotrophic hypothesis of depression [10]There are several additional growth factors that also havebeen implicated in neurogenesis depression and treatmentresponse such as insulin-like growth factor-1 (IGF-1) vascu-lar endothelial growth factor (VEGF) and fibroblast growthfactor-2 (FGF-2) [11]

It was reported that swimming exercise could reversethe chronic unpredictable mild stress (CUMS) induceddepression-like state in rodents [12ndash14] However the resultsare inconsistent and the underlying mechanisms are farfrom fully understood [15] Since neurotrophic factors aresuggested to exert a major function in the antidepressanteffects of exercise the main objective of the present study wasto further establish the therapeutic role of swimming exer-cise in depression and systematically evaluate the potentialneurotrophic factors that were involved in the antidepressiveeffects of the exercise paradigmThe expression of biomarkersof cell survival including the antiapoptotic protein Bcl-xl andthe proapoptotic protein Bax was also assessed [16]

2 Materials and Methods

21 Animals Experiments were carried out with maleSprague-Dawley rats (250ndash280 g) supplied by the Experi-mental Animal Center of the Second Xiangya Hospital Therats were housed at 22ndash25∘C and humidity 50ndash60 with a12 h light-dark cycle and had free access to commercial ratchow and water except when they were submitted to CUMSAll animal use procedures were carried out in accordancewith theRegulations of ExperimentalAnimalAdministrationissued by the State Committee of Science and Technology ofthe Peoplersquos Republic of China with the approval of the EthicsCommittee in our university

22 CUMS Procedure and Exercise Protocol The rats wererandomly divided into four groups (119899 = 8) Control groupExercised group Stressed group and Stressed + Exercisedgroup While the rats in Control group were undisturbedthe Exercised group was trained in a progressively increasingmoderate swimming protocol as previously reported withminor change [13] Swimming exercise was performed in aplastic water tank (100 cm times 80 cm times 90 cm) at 32 plusmn 1∘C and adepth of 55 cmThe protocol included two phases adaptationand training In the adaptive phase the rats swam 20minper day for 6 days The adaptation was aimed at reducingthe water induced stress without promoting physiologicalalterations in relation to the physical training [17] After restfor one day the training period began from the first dayof the second week Swimming duration was progressively

increased from 20min to 70min per day for 6 days in 1weekThis intensity wasmaintained to the end of the trainingprogram The swimming program lasted 6 days per week fora total of 4 weeks The beneficial effects of the swimmingprotocol have been repeatedly validated by previous reports[13 18]

The Stressed group received a previously establishedweekly stress regime with minor modification [19] Brieflythe CUMS rats were randomly exposed to one of thesestressors per day for 4 weeks and the same stressor was notused in 2 consecutive days cage tilting (45∘) for 24 h waterdeprivation for 24 h finally with 1 h an empty bottle 1min tailclamping physical restraint for 2 hours fasting for 24 h soiledcage for 24 h and 20min noise The Stress + Exercised ratsreceived both CUMS and the exercise protocol As isolationmay cause additional stress to the animals [20] the Stressedand Stressed + Exercised group were housed separately (cagesize 26 times 19 times 15 cm) while four rats in the other two groupsshared one cage (cage size 90 times 45 times 25 cm)

23 Sucrose Preference Test (SPT) SPT is a measure ofstressed-induced anhedonia state a key depressive-likebehavior in rats [19] Prior to SPT all the rats were housedindividually and habituated to 48 h of forced 1 sucrosesolution consumption in two bottles on each side Thenafter 16 h water deprivation we placed two preweightedbottles one containing 1 sucrose solution and anothercontaining tap water to each rat The side (left and right)of the two bottles was randomly placed in order to avoidspatial bias The bottles were weighted again after 1 h and theweight difference was considered to be the rat intake fromeach bottle The preference for sucrose was measured as apercentage of the consumed 1 sucrose solution relative tothe total amount of liquid intake

24 Open Field Test (OFT) OFT is a measure to evaluate thegeneral locomotion and exploratory behavior of rats [12] Aspreviously described the test was conducted between 800and 1200 one day after SPT Each rat was placed at the centreof an apparatus with a square arena (90 cm times 90 cm times 40 cm)The floor of the arena was equally divided into 25 squaresThe locomotor activity was video-taped and the time in thecentre square and number of crossings (squares of crossingswith all paws) were manually counted for 5min Between thetests the apparatus was thoroughly cleaned and dried

25 Real-Time PCR Analysis Total RNA from hippocampuswas isolated usingTrizol reagent (InvitrogenUSA) accordingto the manufacturerrsquos instructions Quantification of mRNAswas performed on Bio-rad Cx96 Detection System (Bio-radUSA) using SYBR green PCR kit (Applied Biosystems USA)and gene-specific primers Each cDNAwas tested in triplicatewith 40 cycles of amplification Relative quantitation forPCR product was normalized to 120573-actin as internal standardThe sequences of gene-specific primers are summarized inTable 1

26 Statistics Statistical analysis was performed with SPSS130 software All values were presented asmeanplusmn SEM Body

Evidence-Based Complementary and Alternative Medicine 3

Table 1 Primer sequences used for the qPCR analysis

Gene (accession no) Sense primer (51015840-31015840) Antisense primer (51015840-31015840) Amplicon lengthBDNF (NM001270630) TACCTGGATGCCGCAAACAT GTAGAAATATTGCTTCAGTTGG 200 bpGDNF (NM019139) CAGAGGGAAAGGTCGCAGAG CGTAGCCCAAACCCAAGTCA 95 bpNT-3 (NM001270868) GGGAGAGATCAAAACCGGC TTGCGACGTTTTGCACT 136 bpNGF (NM001277055) ACATCAAGGGCAAGGAGG GTGAGTCGTGGTGCAGTATG 164 bpFGF-2 (NM019305) TCCATCAAGGGAGTGTGTGC TCCGTGACCGGTAAGTGTTG 139 bpVEGF (NM001110333) TATGTTTGACTGCTGTGGACTTGA CAGGGATGGGTTTGTCGTGT 204 bpIGF-1 (NM001082477) TCAGTTCGTGTGTGGACCAG CACAGCTCCGGAAGCAAC 117 bpNPY (NM012614) TGTTTGGGCATTCTGGCTGAGG TTCTGGGGGCGTTTTCTGTGCT 205 bpVGF (NM030997) GATGAGTTGCCGGACTGG CAACGCGTGATGGAAGTGAC 159 bpBcl-xl (NM001033670) TGTGGCTGGTGTAGTTCTGC CAGAAAAGCATTCCCGAGAG 402 bpBax (NM017059) CTGCAGAGGATGATTGCTGA GATCAGCTCGGGCACTTTAG 174 bp120573-Actin (NM031144) CATCCTGCGTCTGGACCTGG TAATGTCACGCACGATTTCC 116 bp

weight gain was analyzed using a two-way repeated ANOVAanalysis with Stress and Exercise as between subject factorsand time as within subject factor The data of SPT OFTand relative mRNA expression were analyzed by using two-way ANOVA followed by Fisherrsquos least significant difference(LSD) post hoc test The prior level of significance wasestablished at 119875 lt 005

3 Results

31 Behavioral Test and Body Weight Gain Both exerciseand CUMS procedure significantly slowed down the bodyweight growing rate (Figure 1(a)) Stress exposure induced asignificant decrease in sucrose preference Exercise had noeffect on sucrose intake in nonstressed rats but significantlyreversed the decline in the sucrose preference of CUMSrats (Figure 1(b)) The locomotor activity and exploratorybehavior were observed in the OFT (Figures 1(c) and 1(d))4 weeks of CUMS led to a significantly decreased number ofcrossings and increased time spent in centre The Exercisedgroup seems to be more explorative with less time spentin centre and more numbers of crossings The Stressed +Exercised group also showed improved exploratory behaviorcompared with Stressed group Overall the results suggestthat swimming exercise can successfully ameliorate thestress-induced depression-like state in rats

32 Neurotrophic Effects of Swimming Exercise Since neu-rotrophic factors play a major role in the antidepressantaction of exercise we assessed the expression of the neu-rotrophic factors that related to depression in the hip-pocampus As previously reported [21] exercise significantlyinduced the expression of BDNF the mostly documentedbiomarker in depression and reversed the inhibitory effectsof chronic stress (Figure 2(a)) Interestingly stress inducedGDNF (Figure 2(b)) and NT-3 (Figure 2(c)) expressionwhile no influence of exercise on the two neurotrophins wasfound which suggests a potential compensatory adaptativemechanism to CUMS Both NGF (Figure 2(d)) and FGF-2 (Figure 2(e)) were downregulated by CUMS while theswimming protocol also had no effect on their expression

Hippocampal generation of VEGF (Figure 2(f)) and IGF-1 (Figure 2(g)) was not influenced by chronic stress butexercise significantly increased the expression of IGF-1 innon-CUMS rats In parallel to the expression of BDNFthe mRNA levels of its downstream neurotrophic peptidesNPY (Figure 2(h)) andVGF (Figure 2(i)) were synchronouslyinhibited by stress and restored by exercise Chronic stressalso downregulated Bcl-xl mRNA status (Figure 3(a)) andupregulated the expression of Bax (Figure 3(b)) The stress-induced decreased ratio of Bcl-xlBax indicated that theneural cells were shifted toward cell death (Figure 3(c)) How-ever exercise was found to promote Bcl-xl expression andincrease the Bcl-xlBax ratio in non-CUMS rats Meanwhilein Stressed + Exercised group the swimming protocol partlynormalized the stress-induced imbalance between Bcl-xl andBax

4 Discussion

Neural atrophy has been reported in rodent chronic stressmodels and clinical postmortem studies of depressedpatients These findings indicate that depression can beconsidered as mild neurodegenerative disorder and theincreased neural cell loss may contribute to the pathogenesisof depression [22] Recent studies have strengthened therole of the abnormalities in neurotrophic pathways inthe psychopathophysiology of depression It has beenrepeatedly documented that chronic stress and depressionare associated with the abnormal neurotrophic signaling inthe brain [23] Exercise is an effective strategy for depressiontreatment A key mechanism mediating the antidepressantaction of exercise is induction of central and peripherygrowth factor cascades Several classes of neurotrophicfactors are implicated in the neural atrophy of depressionand beneficial effects of exercise Although most work hasfocused on BDNF other growth factors also play a role in thestress-induced neural cell loss

Several lines of evidence from animal and humanresearches converge on the idea that BDNF is essential forhippocampal synaptic plasticity and modulation of depres-sion The exercise-evoked BDNF release has been repeatedly


Nonstress + exerciseStress + exercise

Time (days)

Body

wei

ght (

g)

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

0 9 18 27

200

300

400

500

NonstressStress

(a)

Sucr

ose p

refe

renc

e (

) +

lowastlowast

0

20

40

60

80

100

Nonstress Stress

SedentarySwimming exercise

(b)

lowast ++

lowastlowast

0

5

10

15

20

Tim

e in

cent

re (s

)


Nonstress Stress

(c)

lowast

++

lowastlowast

0

20

40

60

80


Nonstress Stress

Num

ber o

f cro

ssin

gs

(d)

Figure 1 Effect of swimming exercise and CUMS on rat body weight (a) and behavioral changes in sucrose preference test (b) and time spentin centre (c) and number of crossings (d) in open field test Data are means plusmn SEM (119899 = 8) lowast119875 lt 005 lowastlowast119875 lt 001 compared to Control group+

119875 lt 005 ++119875 lt 001 compared to Stressed group

postulated to underlie the antidepressant effects of physicalactivity [6 23 24] In the present study we confirmed theantidepressant effects of the swimming protocol and aspreviously reported the induction of BDNF expression wasalso found in this study suggesting the involvement of BDNFin the amelioration of depression by swimming exerciseInterestingly in parallel to BDNF a synchronous rise of NPYand VGF was also found in the Exercised group The mRNAlevels of NPY and VGF were inhibited in Stressed groupbut were normalized in the Stressed + Exercised group BothNPY and VGF are related to neuronal synaptic remodelingand could be highly induced by BDNF in vitro and in vivo[9 25] As NPY and VGF also actively participate in theneurotrophic hypothesis of depression and could be regulatedby BDNF our data argue that both BDNF and its downstreamneurotrophic peptides NPY and VGF may play a role in theantidepressant effects of exercise

Additionally the effects of swimming exercise and stressexposure on other neurotrophic factors including GDNFNT-3 NGF and FGF-2 were also evaluated in this studyThe exercise protocol had no influence on the four factorsexpression but a significant reduction ofNGF and FGF-2wasfound in the Stressed groupWhile inhibiting NGF and FGF-2 expression it has been reported that exposure to chronicstress upregulated NT-3 and GDNF [26 27] However theresults were inconsistent [28 29] In our study we confirmedthe increase of NT-3 and GDNF which suggests a compen-satory adaptive process to the stress-induced alterations ofneurotrophic signaling in the hippocampus

VEGF and IGF-1 are other two principle growth factorsknown to mediate the effects of exercise on the brain Unlikethe neurotrophins above although both VEGF and IGF-1can be synthesized in the brain peripheral production is themain source of circulating VEGF and IGF-1 Both VEGF and


lowast

++

lowastlowast

00

05

10

15

20

Nonstress Stress

BDN

F m

RNA

expr

essio

n

(a)

lowast

0

1

2

3

Nonstress Stress

GD

NF

mRN

A ex

pres

sion

(b)

lowastlowast

+

0

1

2

3

Nonstress Stress

NT-3

mRN

A ex

pres

sion

(c)

lowastlowast

00

05

10

15

Nonstress Stress

NG

F m

RNA

expr

essio

n

(d)

lowast

00

05

10

15

Nonstress Stress

FGF2

mRN

A ex

pres

sion

(e)

00

05

10

15

Nonstress Stress

VEG

F m

RNA

expr

essio

n

(f)

lowast

00

05

10

15

20

25

Nonstress Stress


IGF-1

mRN

A ex

pres

sion

(g)

lowast

++

lowastlowast

00

05

10

15

20

25

Nonstress Stress

NPY

mRN

A ex

pres

sion


(h)

Figure 2 Continued


lowast

lowast

++

00

05

10

15

20

25


Nonstress Stress

VGF

mRN

A ex

pres

sion

(i)

Figure 2 Effect of swimming exercise and CUMS on the neurotrophic factors expression Data are means plusmn SEM (119899 = 6) lowast119875 lt 005lowastlowast

119875 lt 001 compared to Control group +119875 lt 005 ++119875 lt 001 compared to Stressed group

lowast

lowast

++

Nonstress Stress00

05

10

15

20

Bcl-x

l mRN

A ex

pres

sion


(a)

lowast

+

Nonstress Stress00

05

10

15

20

Bax

mRN

A ex

pres

sion


(b)

++

lowastlowast

lowastlowast

Nonstress Stress0

1

2

3

4

Bcl-x

lBax

ratio


(c)

Figure 3 Effect of swimming exercise and CUMS on the expression of Bcl-xl (a) and Bax (b) and ratio of Bcl-xlBax (c) Data are means plusmnSEM (119899 = 6) lowast119875 lt 005 lowastlowast119875 lt 001 compared to Control group +119875 lt 005 ++119875 lt 001 compared to Stressed group


IGF-1 are essential for exercise stimulated neurogenesis andblocking either VEGF or IGF-1 signaling prevents exercise-induced neurogenesis in the hippocampus [30 31] Howeverthe evidence of the influence of exercise on central generationof VEGF and IGF-1 is limited In our study we found thatexercise only increased the hippocampal IGF-1 expressionwithout any alterations of VEGF Nevertheless the significantchange was not found between Stressed and Stressed +Exercised group implying that the hippocampal productionof IGF-1 does not play a key role in the antidepressanteffects of swimming exercise Since both VEGF and IGF-1 are increased in the periphery by exercise and can crossthe blood-brain-barrier to enter the brain [32 33] our dataindicate that the beneficial effects of exercise appear to bemediated by the periphery genesis of the two factors but notthrough the influence on paracrine effects of VEGF and IGF-1in the hippocampus

The neural apoptosis process is programmed and con-trolled by the cellular balance between antiapoptotic Bcl-xl and proapoptotic Bax expression As previously reportedchronic stress downregulated the expression of Bcl-xl andupregulated Bax status [34 35] The altered balance indicatesthat the neural cell is shifted toward apoptosis The beneficialeffect of swimming exercise on neural survival was furthervalidated by the restored balance between Bcl-xl and BaxThe enhanced expression of Bcl-xl was also found in thenon-CUMS rats after the exercise protocol suggesting thatswimming exercise can not only reverse the apoptotic effectof stress but also improve the basal neural cell survival

5 Conclusions

Collectively since the neurotrophic signaling can regulateneural survival and Bcl-xlBax expression and the reversedeffects of swimming exercise on the stressinduced neu-rotrophic alterations were only found in BDNF and itsdownstream peptides (NPY and VGF) expression our datasuggests that the neurotrophic benefits of swimming exercisein the hippocampus are mainly through the BDNF signalingpathway and NPY and VGF also synergetically play a role inthe antidepressant action of swimming exercise

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This research was supported by Hunan Provincial InnovationFoundation for Postgraduate (no CX2013B099) andNationalNatural Science Foundation of China (no 81101001)

References

[1] G Masi and P Brovedani ldquoThe hippocampus neurotrophicfactors and depression possible implications for the pharma-cotherapy of depressionrdquoCNSDrugs vol 25 no 11 pp 913ndash9312011

[2] H Eyre and B T Baune ldquoNeuroimmunological effects ofphysical exercise in depressionrdquo Brain Behavior and Immunityvol 26 no 2 pp 251ndash266 2012

[3] M Llorens-Martın I Torres-Aleman and J L Trejo ldquoGrowthfactors as mediators of exercise actions on the brainrdquo Neuro-Molecular Medicine vol 10 no 2 pp 99ndash107 2008

[4] C W Cotman N C Berchtold and L-A Christie ldquoExercisebuilds brain health key roles of growth factor cascades andinflammationrdquo Trends in Neurosciences vol 30 no 9 pp 464ndash472 2007

[5] R S Duman and G K Aghajanian ldquoSynaptic dysfunction indepression potential therapeutic targetsrdquo Science vol 338 no6103 pp 68ndash72 2012

[6] L Marais D J Stein and W M U Daniels ldquoExercise increasesBDNF levels in the striatum and decreases depressive-likebehavior in chronically stressed ratsrdquo Metabolic Brain Diseasevol 24 no 4 pp 587ndash597 2009

[7] M P Mattson ldquoEnergy intake and exercise as determinantsof brain health and vulnerability to injury and diseaserdquo CellMetabolism vol 16 no 6 pp 706ndash722 2012

[8] M Heilig ldquoThe NPY system in stress anxiety and depressionrdquoNeuropeptides vol 38 no 4 pp 213ndash224 2004

[9] S Thakker-Varia and J Alder ldquoNeuropeptides in depressionrole of VGFrdquoBehavioural Brain Research vol 197 no 2 pp 262ndash278 2009

[10] W N Marsden ldquoSynaptic plasticity in depression Molec-ular cellular and functional correlatesrdquo Progress in Neuro-Psychopharmacology and Biological Psychiatry vol 43 pp 168ndash184 2013

[11] M Banasr JMDwyer and R S Duman ldquoCell atrophy and lossin depression reversal by antidepressant treatmentrdquo CurrentOpinion in Cell Biology vol 23 no 6 pp 730ndash737 2011

[12] W Liu and C Zhou ldquoCorticosterone reduces brain mito-chondrial function and expression of mitofusin BDNF indepression-like rodents regardless of exercise preconditioningrdquoPsychoneuroendocrinology vol 37 no 7 pp 1057ndash1070 2012

[13] X Liu L J Yang S J Fan H Jiang and F Pan ldquoSwim-ming exercise effects on the expression of HSP70 and iNOSin hippocampus and prefrontal cortex in combined stressrdquoNeuroscience Letters vol 476 no 2 pp 99ndash103 2010

[14] A R Sigwalt H Budde I Helmich et al ldquoMolecular aspectsinvolved in swimming exercise training reducing anhedonia ina rat model of depressionrdquo Neuroscience vol 192 pp 661ndash6742011

[15] Y Zhao L-J Liu C Wang and S-X Li ldquoSwimming exercisemay not alleviate the depressive-like behaviors and circadianalterations of neuroendocrine induced by chronic unpre-dictable mild stress in ratsrdquo Neurology Psychiatry and BrainResearch vol 18 no 4 pp 202ndash207 2012

[16] Y Wang Z Xiao X Liu and M Berk ldquoVenlafaxine mod-ulates depression-induced behaviour and the expression ofBax mRNA and Bcl-xl mRNA in both hippocampus andmyocardiumrdquo Human Psychopharmacology vol 26 no 2 pp95ndash101 2011

[17] R V L Contarteze F D BManchado C A Gobatto andM AR de Mello ldquoStress biomarkers in rats submitted to swimmingand treadmill running exercisesrdquoComparative Biochemistry andPhysiology A Molecular and Integrative Physiology vol 151 no3 pp 415ndash422 2008

[18] W Liu H Sheng Y Xu Y Liu J Lu and X Ni ldquoSwimmingexercise ameliorates depression-like behavior in chronically


stressed rats relevant to proinflammatory cytokines and IDOactivationrdquo Behavioural Brain Research vol 242 no 1 pp 110ndash116 2013

[19] P Jiang W-Y Zhang H-D Li H-L Cai Y-P Liu and L-Y Chen ldquoStress and vitamin D altered vitamin D metabolismin both the hippocampus and myocardium of chronic unpre-dictable mild stress exposed ratsrdquo Psychoneuroendocrinologyvol 38 no 10 pp 2091ndash2098 2013

[20] P C Casarotto and R Andreatini ldquoRepeated paroxetine treat-ment reverses anhedonia induced in rats by chronic mild stressor dexamethasonerdquo European Neuropsychopharmacology vol17 no 11 pp 735ndash742 2007

[21] Z Radak A Toldy Z Szabo et al ldquoThe effects of trainingand detraining on memory neurotrophins and oxidative stressmarkers in rat brainrdquo Neurochemistry International vol 49 no4 pp 387ndash392 2006

[22] Z Z Meng J X Chen Y M Jiang and H T Zhang ldquoEffectof xiaoyaosan decoction on learning and memory deficit inrats induced by chronic immobilization stressrdquo Evidence-BasedComplementary and Alternative Medicine vol 2013 Article ID297154 8 pages 2013

[23] Ł R Drzyzga AMarcinowska and E Obuchowicz ldquoAntiapop-totic and neurotrophic effects of antidepressants a review ofclinical and experimental studiesrdquo Brain Research Bulletin vol79 no 5 pp 248ndash257 2009

[24] J Maniam and M J Morris ldquoVoluntary exercise and palat-able high-fat diet both improve behavioural profile and stressresponses in male rats exposed to early life stress role ofhippocampusrdquo Psychoneuroendocrinology vol 35 no 10 pp1553ndash1564 2010

[25] J Alder SThakker-Varia D A Bangasser et al ldquoBrain-derivedneurotrophic factor-induced gene expression reveals novelactions of VGF in hippocampal synaptic plasticityrdquo Journal ofNeuroscience vol 23 no 34 pp 10800ndash10808 2003

[26] Y Bian Z Pan ZHouCHuangW Li andB Zhao ldquoLearningmemory and glial cell changes following recovery from chronicunpredictable stressrdquo Brain Research Bulletin vol 88 no 5 pp471ndash476 2012

[27] T Lee J Saruta K Sasaguri S Sato andK Tsukinoki ldquoAllowinganimals to bite reverses the effects of immobilization stresson hippocampal neurotrophin expressionrdquo Brain Research vol1195 pp 43ndash49 2008

[28] Q Liu H-Y Zhu B Li Y-Q Wang J Yu and G-CWu ldquoChronic clomipramine treatment restores hippocampalexpression of glial cell line-derived neurotrophic factor in a ratmodel of depressionrdquo Journal of Affective Disorders vol 141 no2-3 pp 367ndash372 2012

[29] K Y Zhu Q-Q Mao S-P Ip et al ldquoA standardized chineseherbal decoction kai-xin-san restores decreased levels of neu-rotransmitters and neurotrophic factors in the brain of chronicstress-induced depressive ratsrdquo Evidence-Based Complementaryand Alternative Medicine vol 2012 Article ID 149256 13 pages2012

[30] T Kiuchi H Lee and T Mikami ldquoRegular exercise curesdepression-like behavior via VEGF-Flk-1 signaling in chroni-cally stressed micerdquo Neuroscience vol 207 pp 208ndash217 2012

[31] J L Trejo M V LLorens-Martın and I Torres-Aleman ldquoTheeffects of exercise on spatial learning and anxiety-like behaviorare mediated by an IGF-I-dependent mechanism related to hip-pocampal neurogenesisrdquo Molecular and Cellular Neurosciencevol 37 no 2 pp 402ndash411 2008

[32] K Fabel K Fabel B Tam et al ldquoVEGF is necessary for exercise-induced adult hippocampal neurogenesisrdquo European Journal ofNeuroscience vol 18 no 10 pp 2803ndash2812 2003

[33] J L Trejo E Carro and I Torres-Aleman ldquoCirculating insulin-like growth factor I mediates exercise-induced increases in thenumber of new neurons in the adult hippocampusrdquoThe Journalof Neuroscience vol 21 no 5 pp 1628ndash1634 2001

[34] A Bachis M I Cruz R L Nosheny and I MocchettildquoChronic unpredictable stress promotes neuronal apoptosis inthe cerebral cortexrdquo Neuroscience Letters vol 442 no 2 pp104ndash108 2008

[35] T A Kosten M P Galloway R S Duman D S Russell andC DrsquoSa ldquoRepeated unpredictable stress and antidepressantsdifferentially regulate expression of the Bcl-2 family of apoptoticgenes in rat cortical hippocampal and limbic brain structuresrdquoNeuropsychopharmacology vol 33 no 7 pp 1545ndash1558 2008

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both of which can be induced by BDNF and antidepressants[8 9] Accumulating evidence has shown that BDNF wasimplicated in the pathophysiology of depression and theantidepressant action of exercise Nevertheless the role ofits downstream neuropeptides and other neurotrophic fac-tors remains unclear Other neurotrophins including nervegrowth factor (NGF) glia cell-derived neurotrophic factor(GDNF) and neurotrophin-3 (NT-3) are also importantfactors for regulation of neuroplasty and were implied toplay a role in the neurotrophic hypothesis of depression [10]There are several additional growth factors that also havebeen implicated in neurogenesis depression and treatmentresponse such as insulin-like growth factor-1 (IGF-1) vascu-lar endothelial growth factor (VEGF) and fibroblast growthfactor-2 (FGF-2) [11]

It was reported that swimming exercise could reversethe chronic unpredictable mild stress (CUMS) induceddepression-like state in rodents [12ndash14] However the resultsare inconsistent and the underlying mechanisms are farfrom fully understood [15] Since neurotrophic factors aresuggested to exert a major function in the antidepressanteffects of exercise the main objective of the present study wasto further establish the therapeutic role of swimming exer-cise in depression and systematically evaluate the potentialneurotrophic factors that were involved in the antidepressiveeffects of the exercise paradigmThe expression of biomarkersof cell survival including the antiapoptotic protein Bcl-xl andthe proapoptotic protein Bax was also assessed [16]

2 Materials and Methods

21 Animals Experiments were carried out with maleSprague-Dawley rats (250ndash280 g) supplied by the Experi-mental Animal Center of the Second Xiangya Hospital Therats were housed at 22ndash25∘C and humidity 50ndash60 with a12 h light-dark cycle and had free access to commercial ratchow and water except when they were submitted to CUMSAll animal use procedures were carried out in accordancewith theRegulations of ExperimentalAnimalAdministrationissued by the State Committee of Science and Technology ofthe Peoplersquos Republic of China with the approval of the EthicsCommittee in our university

22 CUMS Procedure and Exercise Protocol The rats wererandomly divided into four groups (119899 = 8) Control groupExercised group Stressed group and Stressed + Exercisedgroup While the rats in Control group were undisturbedthe Exercised group was trained in a progressively increasingmoderate swimming protocol as previously reported withminor change [13] Swimming exercise was performed in aplastic water tank (100 cm times 80 cm times 90 cm) at 32 plusmn 1∘C and adepth of 55 cmThe protocol included two phases adaptationand training In the adaptive phase the rats swam 20minper day for 6 days The adaptation was aimed at reducingthe water induced stress without promoting physiologicalalterations in relation to the physical training [17] After restfor one day the training period began from the first dayof the second week Swimming duration was progressively

increased from 20min to 70min per day for 6 days in 1weekThis intensity wasmaintained to the end of the trainingprogram The swimming program lasted 6 days per week fora total of 4 weeks The beneficial effects of the swimmingprotocol have been repeatedly validated by previous reports[13 18]

The Stressed group received a previously establishedweekly stress regime with minor modification [19] Brieflythe CUMS rats were randomly exposed to one of thesestressors per day for 4 weeks and the same stressor was notused in 2 consecutive days cage tilting (45∘) for 24 h waterdeprivation for 24 h finally with 1 h an empty bottle 1min tailclamping physical restraint for 2 hours fasting for 24 h soiledcage for 24 h and 20min noise The Stress + Exercised ratsreceived both CUMS and the exercise protocol As isolationmay cause additional stress to the animals [20] the Stressedand Stressed + Exercised group were housed separately (cagesize 26 times 19 times 15 cm) while four rats in the other two groupsshared one cage (cage size 90 times 45 times 25 cm)

23 Sucrose Preference Test (SPT) SPT is a measure ofstressed-induced anhedonia state a key depressive-likebehavior in rats [19] Prior to SPT all the rats were housedindividually and habituated to 48 h of forced 1 sucrosesolution consumption in two bottles on each side Thenafter 16 h water deprivation we placed two preweightedbottles one containing 1 sucrose solution and anothercontaining tap water to each rat The side (left and right)of the two bottles was randomly placed in order to avoidspatial bias The bottles were weighted again after 1 h and theweight difference was considered to be the rat intake fromeach bottle The preference for sucrose was measured as apercentage of the consumed 1 sucrose solution relative tothe total amount of liquid intake

24 Open Field Test (OFT) OFT is a measure to evaluate thegeneral locomotion and exploratory behavior of rats [12] Aspreviously described the test was conducted between 800and 1200 one day after SPT Each rat was placed at the centreof an apparatus with a square arena (90 cm times 90 cm times 40 cm)The floor of the arena was equally divided into 25 squaresThe locomotor activity was video-taped and the time in thecentre square and number of crossings (squares of crossingswith all paws) were manually counted for 5min Between thetests the apparatus was thoroughly cleaned and dried

25 Real-Time PCR Analysis Total RNA from hippocampuswas isolated usingTrizol reagent (InvitrogenUSA) accordingto the manufacturerrsquos instructions Quantification of mRNAswas performed on Bio-rad Cx96 Detection System (Bio-radUSA) using SYBR green PCR kit (Applied Biosystems USA)and gene-specific primers Each cDNAwas tested in triplicatewith 40 cycles of amplification Relative quantitation forPCR product was normalized to 120573-actin as internal standardThe sequences of gene-specific primers are summarized inTable 1

26 Statistics Statistical analysis was performed with SPSS130 software All values were presented asmeanplusmn SEM Body





3 Results




4 Discussion





Time (days)

Body

wei

ght (

g)

lowast

lowastlowast

lowastlowast

lowastlowast

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0 9 18 27

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500

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(a)

Sucr

ose p

refe

renc

e (

) +

lowastlowast

0

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40

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Nonstress Stress


(b)

lowast ++

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5

10

15

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Tim

e in

cent

re (s

)


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(c)

lowast

++

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Nonstress Stress

Num

ber o

f cro

ssin

gs

(d)







lowast

++

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expr

essio

n

(a)

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sion

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sion

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expr

essio

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mRN

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sion

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VEG

F m

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expr

essio

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IGF-1

mRN

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sion

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lowast

++

lowastlowast

00

05

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25

Nonstress Stress

NPY

mRN

A ex

pres

sion


(h)

Figure 2 Continued


lowast

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++

00

05

10

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25


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VGF

mRN

A ex

pres

sion

(i)



lowast

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l mRN

A ex

pres

sion


(a)

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mRN

A ex

pres

sion


(b)

++

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lowastlowast

Nonstress Stress0

1

2

3

4

Bcl-x

lBax

ratio


(c)





5 Conclusions




Acknowledgments


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















3 Results




4 Discussion





Time (days)

Body

wei

ght (

g)

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

0 9 18 27

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300

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500

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(a)

Sucr

ose p

refe

renc

e (

) +

lowastlowast

0

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(b)

lowast ++

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Tim

e in

cent

re (s

)


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(c)

lowast

++

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ber o

f cro

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(d)







lowast

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BDN

F m

RNA

expr

essio

n

(a)

lowast

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GD

NF

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sion

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sion

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IGF-1

mRN

A ex

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sion

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lowast

++

lowastlowast

00

05

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15

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Nonstress Stress

NPY

mRN

A ex

pres

sion


(h)

Figure 2 Continued


lowast

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++

00

05

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25


Nonstress Stress

VGF

mRN

A ex

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sion

(i)



lowast

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l mRN

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sion


(a)

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mRN

A ex

pres

sion


(b)

++

lowastlowast

lowastlowast

Nonstress Stress0

1

2

3

4

Bcl-x

lBax

ratio


(c)





5 Conclusions




Acknowledgments


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Time (days)

Body

wei

ght (

g)

lowast

lowastlowast

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(a)

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ose p

refe

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) +

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(b)

lowast ++

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Tim

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)


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Num

ber o

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BDN

F m

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n

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sion

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VEG

F m

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n

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IGF-1

mRN

A ex

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sion

(g)

lowast

++

lowastlowast

00

05

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15

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25

Nonstress Stress

NPY

mRN

A ex

pres

sion


(h)

Figure 2 Continued


lowast

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++

00

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25


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VGF

mRN

A ex

pres

sion

(i)



lowast

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l mRN

A ex

pres

sion


(a)

lowast

+

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mRN

A ex

pres

sion


(b)

++

lowastlowast

lowastlowast

Nonstress Stress0

1

2

3

4

Bcl-x

lBax

ratio


(c)





5 Conclusions




Acknowledgments


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















lowast

++

lowastlowast

00

05

10

15

20

Nonstress Stress

BDN

F m

RNA

expr

essio

n

(a)

lowast

0

1

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3

Nonstress Stress

GD

NF

mRN

A ex

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sion

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NT-3

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sion

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lowastlowast

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F m

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expr

essio

n

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FGF2

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A ex

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sion

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VEG

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RNA

expr

essio

n

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Nonstress Stress


IGF-1

mRN

A ex

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sion

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lowast

++

lowastlowast

00

05

10

15

20

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Nonstress Stress

NPY

mRN

A ex

pres

sion


(h)

Figure 2 Continued


lowast

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++

00

05

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20

25


Nonstress Stress

VGF

mRN

A ex

pres

sion

(i)



lowast

lowast

++

Nonstress Stress00

05

10

15

20

Bcl-x

l mRN

A ex

pres

sion


(a)

lowast

+

Nonstress Stress00

05

10

15

20

Bax

mRN

A ex

pres

sion


(b)

++

lowastlowast

lowastlowast

Nonstress Stress0

1

2

3

4

Bcl-x

lBax

ratio


(c)





5 Conclusions




Acknowledgments


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















lowast

lowast

++

00

05

10

15

20

25


Nonstress Stress

VGF

mRN

A ex

pres

sion

(i)



lowast

lowast

++

Nonstress Stress00

05

10

15

20

Bcl-x

l mRN

A ex

pres

sion


(a)

lowast

+

Nonstress Stress00

05

10

15

20

Bax

mRN

A ex

pres

sion


(b)

++

lowastlowast

lowastlowast

Nonstress Stress0

1

2

3

4

Bcl-x

lBax

ratio


(c)





5 Conclusions




Acknowledgments


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















5 Conclusions




Acknowledgments


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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